AU758073B2 - Serum free medium for chondrocyte-like cells - Google Patents
Serum free medium for chondrocyte-like cells Download PDFInfo
- Publication number
- AU758073B2 AU758073B2 AU13804/00A AU1380400A AU758073B2 AU 758073 B2 AU758073 B2 AU 758073B2 AU 13804/00 A AU13804/00 A AU 13804/00A AU 1380400 A AU1380400 A AU 1380400A AU 758073 B2 AU758073 B2 AU 758073B2
- Authority
- AU
- Australia
- Prior art keywords
- serum
- cells
- fgf
- chondrocytes
- medium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Ceased
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0655—Chondrocytes; Cartilage
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/05—Inorganic components
- C12N2500/10—Metals; Metal chelators
- C12N2500/20—Transition metals
- C12N2500/24—Iron; Fe chelators; Transferrin
- C12N2500/25—Insulin-transferrin; Insulin-transferrin-selenium
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/36—Lipids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/38—Vitamins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/105—Insulin-like growth factors [IGF]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/11—Epidermal growth factor [EGF]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/125—Stem cell factor [SCF], c-kit ligand [KL]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/135—Platelet-derived growth factor [PDGF]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/235—Leukemia inhibitory factor [LIF]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/30—Hormones
- C12N2501/38—Hormones with nuclear receptors
- C12N2501/39—Steroid hormones
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Rheumatology (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Developmental Biology & Embryology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Hematology (AREA)
- Immunology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
Serum-free media for growth and proliferation of chondrocytes and mesenchymal stem cells in culture are provided. A serum-free medium for growth of chondrocytes includes a serum-free composition comprising FGF-2, linoleic acid, ascorbic acid, B-mercaptoethanol, transferrin and dexamethasone. Further, the composition comprises EGF, PDGFbb, insulin and albumin. A method for growing chondrocytes in a serum free medium comprising the compostion is also provided. Also provided for mesenchymal stem cell growth, is a serum-free medium which includes a composition comprising FGF-2, LIF, SCF, pantotenate, biotin and selenium and method, therefore.
Description
WO 00/27996 PCT/EP99/08482 SERUM FREE MEDIUM FOR CHONDROCYTE-LIKE CELLS BACKGROUND OF THE INVENTION Bone and cartilage transplantation is an absolute need in reconstruction of bone and cartilage segments in plastic surgery, traumatic surgery or after the removal of neoplastic lesions, etc. Typically, material of human (autologous, from donors or from cadavers) or animal origin has been used for this purpose. Given the increased demand from clinicians for transplant tissues, the increased need for microbial safety in tissue transplantation, the advances in cell biology, cell differentiation and tissue engineering, the concept of rebuilding tissues from autologous or allogeneic cells expanded in vitro has become a growing field in the world of biomedical sciences.
Cellular sources for skeletal repair include chondrocytes and cells committed to the chondrocyte lineage, and mesenchymal stem cells, the former specific for cartilage, the latter multipotential and therefore having the potential to be used to replace bone, cartilage and other tissues.
Mesenchymal stem cells (MSCs) are found in bone marrow as well as in blood, dermis and periosteum. Although these cells are normally present at very low frequencies in bone marrow, these cells can be isolated purified and culturally expanded, for example, as described in U.S. Patent No. 5,486,359.
Typically, the in vitro expansion of chondrocytes and MSCs takes place in culture medium supplemented with bovine serum or optimally with autologous serum from the patient. However, the presence of animal or autologous serum in chondrocyte and MSC cultures has certain disadvantages and limitations in view of the potential therapeutical applications of these cultures.
For example, serum is not the physiological fluid most cells closely contact in tissue in vivo. This is particularly true for chondrocytes that, in SUBSTITUTE SHEET (RULE 26) WO 00/27996 PCT/EP99/08482 2 vivo, are embedded in their avascularized matrix and rely for their own growth and differentiation on various growth factors and cytokines acting in an autocrine/panacrine manner rather than diffusing from the distant bloodstream. Further, there is often high variability between animal serum batches. Extensive serum screening required to select the batch most representative of the in vivo inductive effects can be time-consuming and expensive The preparation of autologous serum from patients is also time consuming and supplies are limited. Animal serum can further potentially carry unknown pathogens with consequent risk of contamination for the patient.
Thus, serum substitutes for culturing cells for potential in vivo therapeutic applications is desirable.
SUMMARY OF THE INVENTION The present invention provides a serum substitute for culturing cells in vitro using well defined factors able to support cell viability, proliferation and differentiation as effectively as serum containing medium. In a preferred embodiment, the cells are articular chondrocytes or mesenchymal stem cells.
In one aspect, the invention comprises a composition for the expansion of chondrocytes, comprising a minimum essential medium, a growth factor, albumin, a steroid, an antioxidant, an iron source, a fatty acid and/or a lipid source, and insulin. In a particularly preferred embodiment, the serum free growth medium for chondrocytes comprises FGF-2 as a growth factor, linoleic acid as the lipid/fatty acid source, ascorbic acid and P-mercaptoethanol as antioxidants, holo- and apo-transferrin as the iron source, and dexamethasone as a steroid. Optional ingredients can include cholesterol, trace metals such as selenium, and vitamins such as biotin and sodium pantotenate.
In another aspect, the invention comprises a composition for the maintenance of mesenchymal stem cells, comprising a growth factor, albumin, SUBSTITUTE SHEET (RULE 26) WO 00/27996 PCT/EP99/08482 3 a steroid, an antioxidant, an iron source, a fatty acid and/or a lipid source, one or more vitamins, one or more trace metals, and IGF-1, in combination with a minimum essential medium. In a particularly preferred embodiment, the serum free growth medium for mesenchymal stem cells comprises FGF-2, LIF and SCF as growth factors, sodium pantotenate and biotin as vitamins, and selenium as a trace metal.
BRIEF DESCRIPTION OF THE DRAWING Figure 1 shows the comparison of articular chondrocyte growth kinetics in medium containing FCS and the serum free medium of the present invention.
DETAILED DESCRIPTION OF THE INVENTION The present invention provides serum free compositions suitable for chondrocyte and mesenchymal stem cell growth and proliferation. The compositions may include in a base minimum essential medium, such as Coon's modified Ham's F-12 medium, the following components as a substitute for serum: i) one or more growth factors or proteins which cause resting cells to undergo cell division and/or differentiation, such as insulin, FGF-2, PDGFbb, EGF, LIF and SCF and IGF-1; ii) one or more steroids such as dexamethasone; iii) one or more sources of lipids and fatty acids, necessary for cell membrane biosynthesis, such as cholesterol and linoleic acid; and iv) an iron source such as transferrin.
FGF-2, PDGFbb and EGF are potent mitogens for cells of mesenchymal origin. Dexamethasone is known to'keep cells in a cycling phase in vitro.
The serum free medium of the present invention may further comprise: i) albumin (preferably of mammalian species) which functions as an aspecific carrier; SUBSTITUTE SHEET (RULE 26) WO 00/27996 PCT/EP99/08482 4 ii) one or more antioxidants such as P-mercaptoethanol; iii) a supplement for coenzyme transport in carboxyl group transfer reactions, such as biotin; iv) trace elements as a supplemental source of metal necessary for electron transport and many metalloenzymes and proteins, such as selenium; v) vitamins, such as biotin and pantotenate; and vi) ascorbic acid, to facilitate organization of the extracellular matrix.
Insulin and dexamethasone are added at the average concentrations usually reported in the literature.
IGF-I, LIF and SCF are present at concentrations in the range from about to about 10 ng/ml; preferably at a concentration of 5 ng/ml. All the other components are included in a range of concentration typically used in cell culture studies.
In a preferred embodiment of the composition suitable for the growth and proliferation of the chondrocytes, the defined components comprise EGF, PDGFbb and FGF-2, ascorbic acid, linoleic acid, human serum albumin (HSA), P-mercaptoethanol, dexamethasone, insulin, human holo- and apotransferrin. In this embodiment, FGF-2, PDGFbb and EGF are present at concentrations in the range of from about 1 to about 10 ng/ml. In a preferred embodiment, FGF-2, PDGFbb and EGF are present at concentrations of from 1 to 2 ng/ml.
In a preferred embodiment of the composition suitable for the growth and proliferation of the mesenchymal stem cells, the defined components comprise EGF, PDGFbb and FGF-2, LIF, SCF, IGF-I, ascorbic acid, cholesterol, HSA, P-mercaptoethanol, dexamethasone, human holo- and apotransferrin, selenium, biotin, sodium pantotenate. FGF-2, PDGFbb and EGF are present at concentrations in the range of from about 5 ng/ml to about ng/ml of each factor. The preferred concentrations of FGF-2, PDGFbb and SUBSTITUTE SHEET (RULE 26) WO 00/27996 PCT/EP99/08482 EGF are 10 ng/ml. FGF-2 alone was found to be the most active factor for maintenance of osteochondrogenic potential in MSCs.
Example 1 Articular cartilage was harvested from the knee joint of young adult human donors. The samples were first cleaned of any adherent muscular, connective or subchondral bone tissues, minced into 1-3mm fragments and rinsed in PBS. Single chondrocytes were then released by repeated enzymatic digestions at 37 0 C with 0.25% trypsin, 400U/ml collagenase I, 1000U/ml collagenase II and 1 mg/ml hyaluronidase. Trypsin was then blocked and removed by rapid and extensive washes in PBS containing soybean trypsin inhibitor. Cells were plated in anchorage-dependent conditions in Coon's modified Ham's F-12 medium supplemented either with 10% fetal calf serum (FCS, control culture) or the following defined components: EGF, PDGFbb and FGF-2, ascorbic acid, linoleic acid, human serum albumin (HSA), Pmercaptoethanol, dexamethasone, insulin, human holo- and apo-transferrin.
Table 1 below shows the preferred amounts of each component. To favor adhesion of the cells in serum free conditions, the dishes were pre-coated with 2% gelatin.
Insulin may not be substituted with IGF-1 in the medium for chondrocytes. Insulin was preferably at a concentration of 5 gg/ml.
Selenium, biotin, sodium pantotenate and cholesterol can be routinely included but are optional.
SUBSTITUTE SHEET (RULE 26) WO 00/27996 PCT/EP99/08482 6 Table 1 Medium Supplement for Serum Free Expansion of Human Chondrocytes INGREDIENT CONCENTRATION (Basal medium: Coon's modified Ham's F12) FGF-2 1 -10 ng/ml PDGFbb 1 10 ng/ml EGF 10 ng/ml Insulin 5 upg/ml Dexamethasone 10- M Ascorbic Acid 50 gg/ml Transferrin 20 50 gg/ml HSA 1% Linoleic Acid 6 gM P-mercaptoethanol 5x10-SM Example 2 Bone marrow sample harvested from the iliac crest of the patient was washed twice with PBS. The nucleated cells were counted using methyl violet and plated at 5x10 6 cells as unfractionated marrow per 10 cm tissue culture dish. For selection and expansion, the cells were maintained in Coon's modified Ham's F12 (F12) supplemented either with 10% FCS and 1 ng/ml FGF-2 (control culture) or the following defined components: EGF, PDGFbb, FGF-2, LIF, SCF, IGF-I, ascorbic acid, cholesterol, HSA, P-mercaptoethanol, dexamethasone, human holo- and apo-transferrin, selenium, biotin and sodium pantotenate. Table 2 below shows the preferred amounts of each component.
To favor adhesion of the MSCs, the cells were first plated for 48 hours in F12 medium supplemented with 10% human serum and 1 ng/ml FGF-2.
Thereafter, the medium was removed and the cells were extensively washed with PBS, and left for an additional 24-48 hours in F12 medium without any SUBSTITUTE SHEET (RULE 26) WO 00/27996 PCT/EP99/08482 7 supplement. The defined mixture of factors was then added to promote cell proliferation.
FGF-2 alone was the most active factor for maintenance of osteochondrogenic potential in mesenchymal stem cells. Selenium, biotin and sodium pantotenate were preferably included for cell viability. LIF and SCF were seen to improve the extent of cell proliferation, in particular in combination with IGF-1.
Table 2 Medium Supplement for Serum Free Expansion of MSCs INGREDIENT CONCENTRATION (Basal medium: Coon's modified Ham's F12) Human serum albumin 1-2% Transferrin (apo/holo) 20 50 pg/ml Ascorbic Acid 50 pg/ml p-mercaptoethanol 5 x 10-M Cholesterol 30 gg/ml Selenium 30 nM Biotin 33 um Na pantotenate 17 4M EGF/PDGF/FGF-2 1 10 ng/ml Dexamethasone IGF-I 5 ng/ml LIF 5 ng/ml SCF 5 ng/ml Studies have shown that PDGFbb by itself increases the osteogenic potential of MSCs when included in the phase of proliferation. This effect was found to be amplified by combining PDGFbb with FGF-2.
SUBSTITUTE SHEET (RULE 26) WO 00/27996 PCT/EP99/08482 8 Example 3 Growth Kinetics of Chondrocvtes At day 0, 5 x 103 first passage cells were plated in each well of a 24-well plate in the presence of FCS. Upon adhesion, the FCS was removed, and the cells were extensively washed with PBS and left for 2-3 days in F12 without supplement to exhaust residual traces of serum. Proliferation was then reinduced by adding either 10% FCS or the mixture of defined components established for chondrocytes. Cell number was evaluated at different days via Thiazolyl blue (MTT) staining. Briefly, culture medium was removed and replaced with 0.5 ml of medium without supplement; then 25 .1 MTT (Sigma, St. Louis, MO) stock solution (5 mg/ml) was added to each culture being assayed. After a 3 hour incubation the medium was removed and the converted dye solubilized with absolute ethanol. Absorbance of converted dye was measured at a wavelength of 570 nm with background subtraction at 670 nm.
The data obtained (see Figure 1) clearly show that the defined medium induces the chondrocytes to proliferate to a rate and extent comparable to those obtained in the presence of FCS.
Example 4 The differentiation potential of the chondrocytes expanded in serum free conditions was tested both in vitro and in vivo. For in vitro assay, the expanded cells were transferred in anchorage-independent conditions and maintained as a pellet culture for 2-4 weeks in the serum free medium previously shown by Johnstone et al. (Johnstone, Hering, Caplan, Goldberg, V.M. and Yoo, J.U. Exp. Cell Res. 238, 265-272, 1998) to induce chondrogenesis of serum expanded MSCs.
For in vivo assay, the expanded cells were implanted for 2 to 8. weeks in athymic mice either as a dense cell suspension or after embeddment in fibrin SUBSTITUTE SHEET (RULE 26) WO 00/27996 PCT/EP99/08482 9 gel (Tissucol). At the term of the assays, the samples were fixed in formalin, embedded in paraffin and sectioned. Serial sections were processed for histological (toluidine blue and alcian blue) analysis and immunohistochemistry with collagen-specific antibodies.
(I
Results indicated that, at variance with chondrocytes expanded in the presence of FCS, the chondrocytes expanded in serum free conditions directly reformed a cartilaginous structure both in vitro and in vivo, which stained metachromatic for toluidine blue, positive for alcian blue and type II collagen, and mostly negative for type I collagen. In contrast, in the case of the expansion in FCS, a total absence of full chondrogensis was observed both in vitro and in vivo; at most, a faint metachromatic staining was detected in some pellet cultures, but they always lacked well defined lacunae and well organized extracellular matrix.
These data illustrate a major advantage of the serum free system that allows chondrogenesis without the requirement of additional culturing in the presence of TGF-P 1 or other factors (Johnstone's inducing conditions). This may be due to the fact that chondrocytes, in nature, are not in contact with serum which may contain elements that inhibit chondrogenesis.
Example The osteogenic potential of MSCs after expansion under serum free defined conditions was tested in vivo by implantation of the expanded cells in athymic mice after adsorption on collagraft.
Several combinations of conditions were tested for bone formation in vivo. For all factor combinations, the medium contained Coon's modified Ham's F-12, dexamethasone, FGF-2, PDGFbb, EGF, transferrin, cholesterol, human serum albumin, biotin, selenium, Na pantotenate and ascorbic acid (concentrations as in Table The combinations tested were 1) insulin; 2) IGF-1; 3) insulin and LIF; 4) insulin and SCF; 5) insulin, LIF and SCF; 6) SUBSTITUTE SHEET (RULE 26)
Claims (1)
14-11-2000 EP 009908482 CLAIMS 1. A serum-free composition for the culture of chondrocytes, comprising FGF-2, linoleic acid,. ascorbic acid, 3-mercaptoethanol, transferrin and dexamethasone. 2. A composition according to claim 1, further comprising EGF, PDGFbb, insulin and albumin. 3. A serum-free composition for the culture of mesenchymal stem cells comprising FGF-2, LIF, SCF, pantotenate, biotin and selenium. 4. A composition according to claim 3, further comprising EGF, PDGFbb, IGF-1, ascorbic acid, cholesterol, albumin, b-mercaptoethanol, dexamethasone, transferrin. A composition according to any preceding claim, further comprising a minimum essential medium. 6. A culture of chondrocytes in a serum-free culture medium containing the composition of claims 1-2. 7. A culture of mesenchymal stem cells in a serum-free culture medium containing the composition of claims 3-4. AMENDED SHEET
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US10764698P | 1998-11-09 | 1998-11-09 | |
US60/107646 | 1998-11-09 | ||
PCT/EP1999/008482 WO2000027996A1 (en) | 1998-11-09 | 1999-11-08 | Serum free medium for chondrocyte-like cells |
Publications (2)
Publication Number | Publication Date |
---|---|
AU1380400A AU1380400A (en) | 2000-05-29 |
AU758073B2 true AU758073B2 (en) | 2003-03-13 |
Family
ID=22317692
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
AU13804/00A Ceased AU758073B2 (en) | 1998-11-09 | 1999-11-08 | Serum free medium for chondrocyte-like cells |
Country Status (10)
Country | Link |
---|---|
US (2) | US6617159B1 (en) |
EP (1) | EP1131407B1 (en) |
JP (1) | JP2002529071A (en) |
AT (1) | ATE342348T1 (en) |
AU (1) | AU758073B2 (en) |
CA (1) | CA2348687A1 (en) |
DE (1) | DE69933579D1 (en) |
IL (2) | IL142914A0 (en) |
RU (1) | RU2272839C2 (en) |
WO (1) | WO2000027996A1 (en) |
Families Citing this family (84)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
TR200401091T4 (en) * | 1999-12-28 | 2004-06-21 | Iso Tis N.V. | Cell culture medium containing growth factors and L-glutamine |
JP2004531256A (en) | 2001-04-24 | 2004-10-14 | バクシュ,ドロレス | Progenitor cell populations, their proliferation and the growth of non-hematopoietic cell types and tissues therefrom |
FR2828401B1 (en) * | 2001-08-10 | 2003-11-07 | Oreal | COSMETIC OR DERMATOLOGICAL COMPOSITION COMPRISING AN ASSOCIATION BETWEEN IGF1 AND / OR A MIMETIC COMPOUND OF IGF1, AND ASCORBIC ACID AND / OR AT LEAST ONE OF ITS DERIVATIVES |
JP2003052360A (en) * | 2001-08-20 | 2003-02-25 | Japan Science & Technology Corp | Method for culturing mesenchymal stem cell using extracellular substrate of basement membrane cell |
EP1463800A4 (en) | 2001-12-07 | 2006-04-26 | Geron Corp | Chondrocyte precursors derived from human embryonic stem cells |
US20070141036A1 (en) * | 2002-01-09 | 2007-06-21 | Alberto Gorrochategui Barrueta | Composition and procedure for tissue creation, regeneration and repair by a cell-bearing biological implant enriched with platelet concentrate and supplements |
JP2005515777A (en) | 2002-01-25 | 2005-06-02 | ジェンザイム・コーポレーション | Serum-free medium for chondrocytes and use thereof |
US7741116B2 (en) | 2002-03-06 | 2010-06-22 | University Of Cincinnati | Surgical device for skin therapy or testing |
JP4554940B2 (en) * | 2002-04-03 | 2010-09-29 | 直秀 山下 | Medicament containing mesenchymal cells derived from human placenta and method for producing VEGF using the cells |
EP3175858B1 (en) * | 2002-05-22 | 2019-08-21 | Matsutani Chemical Industry Co., Ltd. | Therapeutic use of d-allose for the treatment of liver cancer or skin cancer |
DE10311620A1 (en) * | 2003-03-17 | 2004-10-07 | Biotissue Technologies Gmbh | Cartilage cell culture medium and use thereof |
AU2003903896A0 (en) | 2003-07-28 | 2003-08-07 | Queensland University Of Technology | Skin regeneration system |
WO2005065354A2 (en) * | 2003-12-31 | 2005-07-21 | The Burnham Institute | Defined media for pluripotent stem cell culture |
DK1725656T3 (en) * | 2004-03-05 | 2014-01-13 | John E Davies | Serum-free suspension culture system for mesenchymal progenitor cells |
WO2005099758A2 (en) * | 2004-04-17 | 2005-10-27 | The Board Of Trustees The Leland Standford Junior University | Injectable bioartificial tissue matrix |
JP2007536935A (en) * | 2004-05-14 | 2007-12-20 | ベクトン・ディキンソン・アンド・カンパニー | Cell culture environment for serum-free growth of mesenchymal stem cells |
EP1783208A4 (en) * | 2004-07-06 | 2007-11-14 | Kyowa Hakko Kogyo Kk | Method of producing nerve cell |
US8697139B2 (en) | 2004-09-21 | 2014-04-15 | Frank M. Phillips | Method of intervertebral disc treatment using articular chondrocyte cells |
JP4646600B2 (en) * | 2004-11-02 | 2011-03-09 | オリンパス株式会社 | Method for culturing mesenchymal stem cells |
JP2006136281A (en) * | 2004-11-15 | 2006-06-01 | Olympus Corp | Medium and method for culturing mesenchymal stem cell |
EP1820852A4 (en) * | 2004-11-19 | 2007-11-14 | Jms Co Ltd | Human serum for cell culture |
US7972767B2 (en) | 2005-05-09 | 2011-07-05 | Olympus Corporation | Method for culturing mesenchymal stem cell and method for producing biological tissue prosthesis |
US20090169523A1 (en) | 2005-06-13 | 2009-07-02 | Catherine Verfaillie | Hsc self-renewal |
DE602006019618D1 (en) | 2005-08-23 | 2011-02-24 | Oriental Yeast Co Ltd | TECHNOLOGY FOR CULTIVATING A MESENCHYMAL STEM CELL USING LAMININ-5 |
JP4921083B2 (en) * | 2005-09-13 | 2012-04-18 | タカラバイオ株式会社 | Serum-free medium for retrovirus production |
US7989205B2 (en) * | 2005-10-06 | 2011-08-02 | American Cryostem Corporation | Cell culture media, kits and methods of use |
DE102005054577A1 (en) * | 2005-11-16 | 2007-05-24 | Cognis Ip Management Gmbh | Use of esters of unsaturated, physiologically active fatty acids as nutrient media for cell cultures |
KR101107835B1 (en) | 2006-01-13 | 2012-02-09 | 가부시키가이샤 투셀 | Culture Medium Additive for Use in Serum-Free Culturing of Animal Cell, Kit, and Use Thereof |
ITTO20060282A1 (en) * | 2006-04-14 | 2007-10-15 | Univ Degli Studi Torino | MEDIUM OF CULTURE AND PHARMACEUTICAL COMPOSITION FOR THE REGENERATION OF THE RELATIVE PAPER FABRIC PROCEDURE RELATED TO USES AND PRODUCTS |
WO2007149328A1 (en) * | 2006-06-20 | 2007-12-27 | Genzyme Corporation | Serum-free media and their uses for chondrocyte expansion |
EP1900810A1 (en) * | 2006-09-15 | 2008-03-19 | Celogos | Method of extracting and selecting cells |
JP2008099662A (en) | 2006-09-22 | 2008-05-01 | Institute Of Physical & Chemical Research | Method for culturing stem cell |
US7951593B2 (en) * | 2007-03-20 | 2011-05-31 | Universite Rene Descartes-Paris V | Culture medium for gingival fibroblasts |
US8114668B2 (en) * | 2007-05-14 | 2012-02-14 | Cardiac Pacemakers, Inc. | Composition for cold storage of stem cells |
ITMI20071421A1 (en) * | 2007-07-16 | 2009-01-17 | Fond Irccs Istituto Di Ricove | CULTURE TO EXPAND EX-LIVE STEM CELLS |
CN101821383B (en) * | 2007-09-05 | 2013-12-18 | 中国医学科学院基础医学研究所 | Culture medium and method for in vitro culturing human adult primary mesenchymal stem cells on large scale, primary mesenchymal stem cells obtained by method, uses thereof |
AU2009205886B2 (en) | 2008-01-18 | 2015-08-27 | Katholieke Universiteit Leuven | Stem cell aggregates and methods for making and using |
US20100015710A1 (en) * | 2008-04-25 | 2010-01-21 | Sunghoon Jung | Methods and Compositions for Isolating, Maintaining and Serially Expanding Human Mesenchymal Stem Cells |
CA2729121C (en) * | 2008-06-30 | 2019-04-09 | Centocor Ortho Biotech Inc. | Differentiation of pluripotent stem cells |
US20110212523A1 (en) * | 2008-11-11 | 2011-09-01 | Yukio Kato | Differentiation-inducing culture medium additive and use thereof |
CA2768573C (en) | 2009-07-21 | 2020-09-15 | Abt Holding Company | A method for constructing a cell bank and a method for drug discovery |
AU2010276201B2 (en) | 2009-07-21 | 2013-10-17 | Abt Holding Company | Use of stem cells to reduce leukocyte extravasation |
SG10201403202XA (en) * | 2009-07-23 | 2014-08-28 | Agency Science Tech & Res | Pre-natal mesenchymal stem cells |
EP4166652A1 (en) * | 2009-11-12 | 2023-04-19 | Technion Research & Development Foundation Ltd. | Culture media, cell cultures and methods of culturing pluripotent stem cells in an undifferentiated state |
JP2013520509A (en) | 2010-02-25 | 2013-06-06 | エイビーティー ホールディング カンパニー | Regulation of angiogenesis |
IL282662B2 (en) | 2010-02-25 | 2024-01-01 | Univ Case Western Reserve | Modulation of macrophage activation |
CN102791276B (en) | 2010-03-10 | 2015-03-04 | 智再如股份有限公司 | Cell preparation containing mesenchymal stem cells, and method for producing same |
US9029145B2 (en) | 2010-04-08 | 2015-05-12 | The University Court Of The University Of Edinburgh | Chondrogenic progenitor cells, protocol for derivation of cells and uses thereof |
WO2011143415A1 (en) | 2010-05-12 | 2011-11-17 | Abt Holding Company | Modulation of splenocytes in cell therapy |
US9090878B2 (en) | 2010-06-17 | 2015-07-28 | Katholieke Universiteit Leuven | Methods for differentiating cells into hepatic stellate cells and hepatic sinusoidal endothelial cells, cells produced by the methods, and methods for using the cells |
SG10201506664TA (en) | 2010-08-24 | 2015-10-29 | Univ Minnesota | Non-static suspension culture of cell aggregates |
EP2625263B1 (en) | 2010-10-08 | 2020-03-11 | Terumo BCT, Inc. | Configurable methods and systems of growing and harvesting cells in a hollow fiber bioreactor system |
WO2012104731A2 (en) | 2011-01-31 | 2012-08-09 | Katholieke Universiteit Leuven | Methods for making cells with an extra-embryonic endodermal precursor phenotype |
JP6182135B2 (en) | 2011-06-06 | 2017-08-16 | レゲネシス ベーフェーベーアー | Amplification of stem cells in a hollow fiber bioreactor |
RU2461623C1 (en) * | 2011-06-20 | 2012-09-20 | Борис Карпович Гаврилюк | Method for cartilage cell culture |
EP2737056B1 (en) | 2011-07-29 | 2018-03-07 | Cellular Dynamics International, Inc. | Metabolic maturation in stem cell-derived tissue cells |
US20140328811A1 (en) | 2011-08-01 | 2014-11-06 | Alnylam Pharmaceuticals, Inc. | Method for improving the success rate of hematopoietic stem cell transplants |
GB201119335D0 (en) | 2011-11-09 | 2011-12-21 | Univ Leuven Kath | Hepatitis virus infectable stem cells |
US9321995B2 (en) * | 2012-12-20 | 2016-04-26 | Suzhou Biowisetech Co., Ltd. | Stem cell culture medium and its applications as well as a stem cell culture method |
WO2014146057A2 (en) * | 2013-03-15 | 2014-09-18 | Cmr Technologies, Llc | Mesenchymal stem cells |
CN115177637A (en) | 2013-04-12 | 2022-10-14 | 休斯顿卫理公会医院 | Improvements in organs for transplantation |
ES2753323T3 (en) | 2013-04-30 | 2020-04-08 | Univ Leuven Kath | Cell therapy for myelodysplastic syndromes |
JP6512759B2 (en) | 2013-08-19 | 2019-05-15 | 国立研究開発法人国立循環器病研究センター | Method of producing amniotic mesenchymal cell composition, method of cryopreservation, and therapeutic agent |
SG11201603407VA (en) * | 2013-11-04 | 2016-05-30 | Isopogen Pty Ltd | Cell culture method |
JP6633522B2 (en) | 2013-11-16 | 2020-01-22 | テルモ ビーシーティー、インコーポレーテッド | Cell growth in bioreactors |
CN103805562B (en) * | 2014-01-13 | 2015-08-05 | 章毅 | Cultivate the serum free medium of placenta mesenchyma stem cell |
US11008547B2 (en) | 2014-03-25 | 2021-05-18 | Terumo Bct, Inc. | Passive replacement of media |
JP6723562B2 (en) * | 2014-05-23 | 2020-07-15 | ガルシア,ホアン パズ | Formulations for the regeneration of bone, cartilage, teeth, and periodontal tissue, and treatment of tumors and cysts |
US9669074B2 (en) * | 2014-05-23 | 2017-06-06 | Juan Paz Garcia | Formulation for regeneration of bone, cartilage, teeth, and periodontium and treatment of tumors and cysts |
US9433629B2 (en) | 2014-05-23 | 2016-09-06 | Juan Paz Garcia | Formulation for regeneration of bone, cartilage, teeth, and periodontium and treatment of tumors and cysts |
JP6830059B2 (en) | 2014-09-26 | 2021-02-17 | テルモ ビーシーティー、インコーポレーテッド | Scheduled cell feeding |
CA2977354C (en) * | 2014-11-14 | 2021-09-21 | Regenesis Science Co., Ltd. | Method for serum-free culture of chondrocytes and serum-free culture medium |
AU2016227607B2 (en) * | 2015-03-04 | 2021-09-30 | Mesoblast International Sarl | Cell culture method for mesenchymal stem cells |
WO2017004592A1 (en) | 2015-07-02 | 2017-01-05 | Terumo Bct, Inc. | Cell growth with mechanical stimuli |
SG11201806245TA (en) | 2016-01-21 | 2018-08-30 | Abt Holding Co | Stem cells for wound healing |
EP3464565A4 (en) | 2016-05-25 | 2020-01-01 | Terumo BCT, Inc. | Cell expansion |
US11104874B2 (en) | 2016-06-07 | 2021-08-31 | Terumo Bct, Inc. | Coating a bioreactor |
US11685883B2 (en) | 2016-06-07 | 2023-06-27 | Terumo Bct, Inc. | Methods and systems for coating a cell growth surface |
US11180733B2 (en) | 2016-07-04 | 2021-11-23 | Agency For Science, Technology And Research | Method of generating mesenchymal stem cells and uses thereof |
US11624046B2 (en) | 2017-03-31 | 2023-04-11 | Terumo Bct, Inc. | Cell expansion |
CN117247899A (en) | 2017-03-31 | 2023-12-19 | 泰尔茂比司特公司 | cell expansion |
AU2018348712B2 (en) | 2017-10-13 | 2024-05-30 | Boehringer Ingelheim International Gmbh | Perfusion medium |
JP7372518B2 (en) * | 2019-06-27 | 2023-11-01 | 国立大学法人山口大学 | Method for culturing bone marrow-derived mesenchymal stem cells |
WO2021158103A1 (en) * | 2020-02-03 | 2021-08-12 | Mosa Meat B.V. | Serum-free medium for culturing a bovine progenitor cell. |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998032333A1 (en) * | 1996-12-06 | 1998-07-30 | Osiris Therapeutics, Inc. | Improved chondrogenic differentiation of human mesenchymal stem cells |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5902741A (en) * | 1986-04-18 | 1999-05-11 | Advanced Tissue Sciences, Inc. | Three-dimensional cartilage cultures |
US5405772A (en) | 1993-06-18 | 1995-04-11 | Amgen Inc. | Medium for long-term proliferation and development of cells |
US5908782A (en) * | 1995-06-05 | 1999-06-01 | Osiris Therapeutics, Inc. | Chemically defined medium for human mesenchymal stem cells |
WO1996040866A1 (en) * | 1995-06-07 | 1996-12-19 | Novartis Ag | Serum-free media for primitive hematopoietic cells and methods of use thereof |
EP0891419A4 (en) * | 1996-03-12 | 2000-03-01 | Life Technologies Inc | Hematopoietic cell culture nutrient supplement |
EP0920490A2 (en) * | 1996-07-25 | 1999-06-09 | Genzyme Corporation | Chondrocyte media formulations and culture procedures |
-
1999
- 1999-11-08 AT AT99971844T patent/ATE342348T1/en not_active IP Right Cessation
- 1999-11-08 US US09/831,161 patent/US6617159B1/en not_active Expired - Fee Related
- 1999-11-08 AU AU13804/00A patent/AU758073B2/en not_active Ceased
- 1999-11-08 EP EP99971844A patent/EP1131407B1/en not_active Expired - Lifetime
- 1999-11-08 WO PCT/EP1999/008482 patent/WO2000027996A1/en active IP Right Grant
- 1999-11-08 DE DE69933579T patent/DE69933579D1/en not_active Expired - Fee Related
- 1999-11-08 IL IL14291499A patent/IL142914A0/en active IP Right Grant
- 1999-11-08 CA CA002348687A patent/CA2348687A1/en not_active Abandoned
- 1999-11-08 RU RU2001116126/13A patent/RU2272839C2/en not_active IP Right Cessation
- 1999-11-08 JP JP2000581163A patent/JP2002529071A/en active Pending
-
2001
- 2001-05-01 IL IL142914A patent/IL142914A/en not_active IP Right Cessation
-
2003
- 2003-07-08 US US10/614,191 patent/US7109032B2/en not_active Expired - Fee Related
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998032333A1 (en) * | 1996-12-06 | 1998-07-30 | Osiris Therapeutics, Inc. | Improved chondrogenic differentiation of human mesenchymal stem cells |
Non-Patent Citations (1)
Title |
---|
QUARTO R ET AL, ENDOCRINOLOGY, 1997, 138 (11):4966-4976 * |
Also Published As
Publication number | Publication date |
---|---|
DE69933579D1 (en) | 2006-11-23 |
IL142914A (en) | 2006-07-05 |
CA2348687A1 (en) | 2000-05-18 |
JP2002529071A (en) | 2002-09-10 |
US6617159B1 (en) | 2003-09-09 |
IL142914A0 (en) | 2002-04-21 |
EP1131407A1 (en) | 2001-09-12 |
AU1380400A (en) | 2000-05-29 |
RU2272839C2 (en) | 2006-03-27 |
US7109032B2 (en) | 2006-09-19 |
EP1131407B1 (en) | 2006-10-11 |
WO2000027996A1 (en) | 2000-05-18 |
US20050090002A1 (en) | 2005-04-28 |
ATE342348T1 (en) | 2006-11-15 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
AU758073B2 (en) | Serum free medium for chondrocyte-like cells | |
EP1988159B1 (en) | Additive for culture medium for use in serum-free culture of animal cell, kit, and use of the additive or kit | |
US6709864B1 (en) | Adipogenic differentiation of human mesenchymal stem cells | |
US6730314B2 (en) | Culturing encapsulated chondrocytes under reduced oxygen partial pressure to produce cartilage implants | |
Broad et al. | Growth and adipose differentiation of sheep preadipocyte fibroblasts in serum‐free medium | |
AU742638B2 (en) | Improved chondrogenic differentiation of human mesenchymal stem cells | |
CN101412985A (en) | Serum-free medium for in vitro cultivation and amplification of mesenchymal stem cells | |
WO2021254296A1 (en) | Bioactive substance composition, serum-free culture medium comprising the composition, and uses thereof | |
KR20060076781A (en) | Cell culture media | |
US20080187518A1 (en) | Production of Osteoclasts from Adipose Tissues | |
US20230117670A1 (en) | Bioactive substance composition, serum-free medium comprising the composition, and uses thereof | |
Webber et al. | Serum‐free culture of rabbit meniscal fibrochondrocytes: Proliferative response | |
CANALIS et al. | Effect of partially purified bone morphogenetic protein on DNA synthesis and cell replication in calvarial and fibroblast cultures. | |
Hendriks et al. | Effect of stratified culture compared to confluent culture in monolayer on proliferation and differentiation of human articular chondrocytes | |
KR20120057784A (en) | Composition for Enhancing Stability of Stem Cells | |
O'Keefe et al. | Countercurrent centrifugal elutriation. High-resolution method for the separation of growth-plate chondrocytes. | |
KR100394430B1 (en) | Medium for culturing human cell comprising human serum and method for culturing human cell using same | |
SHIMOMURA et al. | Cultured growth cartilage cells. | |
US20030026786A1 (en) | Chondrogenic differentiation of human mesenchymal stem cells | |
JP2007000077A (en) | Method for serum-free culture of adherent animal cell and culture medium for serum-free culture of adherent animal cell | |
US20070004037A1 (en) | Methods and compositions for culturing keratinocytes | |
Suryawan et al. | The primary cell culture system for preadipocytes | |
Collodi et al. | Serum-free media | |
Zamani et al. | Comparison of cartilage specific markers in articular and differentiated chondrocytes in pellet system. | |
CN112410296A (en) | Serum-free cell culture medium |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
FGA | Letters patent sealed or granted (standard patent) |